Imaging and analyzing parameters of small moving objects such as cells

ABSTRACT

Light from an object such as a cell moving through an imaging system is collected and dispersed so that it can be imaged onto a time delay and integration (TDI) detector. The light can be emitted from a luminous object or can be light from a light source that has been scattered or not absorbed by the object or can include a light emission by one or more probes within or on the object. Multiple objects passing through the imaging system can be imaged, producing both scatter images and dispersed images at different locations on one or more TDI detectors.

RELATED APPLICATIONS

This application is a continuation-in-part application based on priorcopending patent application Ser. No. 09/490,478, filed on Jan. 24,2000, still pending the benefit of the filing date of which is herebyclaimed under 35 U.S.C. §120.

FIELD OF THE INVENTION

This invention generally relates to imaging moving objects or particlesfor purposes of analysis and detection, and more specifically, to asystem and method for determining and analyzing the morphology of movingobjects, such as cells, and for detecting the presence and compositionof Fluorescence In-Situ Hybridization (FISH) probes within cells.

BACKGROUND OF THE INVENTION

There are a number of biological and medical applications that arecurrently impractical due to limitations in cell and particle analysistechnology. Examples of such biological applications include battlefield monitoring of known airborne toxins, as well as the monitoring ofcultured cells to detect the presence of both known and unknown toxins.Medical applications include non-invasive prenatal genetic testing androutine cancer screening via the detection and analysis of rare cells(i.e., low rate of occurrence) in peripheral blood. All of theseapplications require an analysis system with the following principalcharacteristics:

1. high speed measurement;

2. the ability to process very large or continuous samples;

3. high spectral resolution and bandwidth;

4. good spatial resolution;

5. high sensitivity; and

6. low measurement variation.

In prenatal testing, the target cells are fetal cells that cross theplacental barrier into the mother's bloodstream. In cancer screening,the target cells are sloughed into the bloodstream from nascentcancerous tumors. In both of these applications of this technology, thetarget cells may be present in the blood at concentrations of one tofive cells per billion. This concentration yields approximately 20-100target cells in a typical 20 ml blood sample. The extreme rarity of thetargeted cells demands that any detection and analysis system employedin these applications be capable of processing an enriched sample ofapproximately 100 million cells within a few hours, corresponding to aminimum throughput of 10,000 cells per second. Cell processing includesthe determination of cellular morphology parameters such as overallsize, nuclear size, nuclear shape, and optical density, the detectionand characterization of numerous fluorescent markers and FISH probes,the quantification of the total amount of DNA in the nucleus, and thedetection of other cellular components such as fetal hemoglobin. Toaccomplish these processing tasks, the system must be able to collectcell images with a spatial resolution of approximately 1 micron.Likewise, the system must have high spectral resolution and bandwidth todifferentiate four or more fluorescent colors. Since some probes maylabel important cellular features with only a few thousand fluorescentmolecules, the system must have high sensitivity and good measurementconsistency to differentiate very weak signals.

The predominant research laboratory protocols for non-invasive prenataldiagnosis employ a complex series of process steps that include gradientcentrifugation to remove unnucleated cells, high-speed cell sorting forfetal cell enrichment, and fluorescence microscopy for fetal cellidentification and genetic analysis. These protocols often yield littleor no fetal cells for analysis, because a fraction of the fetal cellsare lost at each step of the protocol. Nevertheless, the protocolscannot be simplified because of limitations in existing analysistechnology. Ideally, fetal cell identification and analysis would beperformed in a few hours by a high-speed cell sorter having thenecessary speed and sample handling capacity. This ideal is not possiblewith conventional systems, because conventional cell sorters lack thenecessary imaging abilities, sensitivity, and repeatability to reliablyidentify fetal cells and enumerate the number and color of FISH probesused to make the diagnosis. Therefore, under current protocols, cellsmust be sorted onto slides and examined using fluorescence microscopy toestablish their fetal origin and make a genetic diagnosis. Thecombination of low fetal cell yields and lengthy processing timesprecludes the clinical application of non-invasive fetal testing withexisting technology.

No technology prior to the present invention incorporates all six of theprincipal characteristics of a viable fetal cell or cancer analysissystem. In the prior art, there have been advances that might be appliedto these applications, but significant limitations still remain.

A paper published by Ong et al. [Anal. Quant. Cytol. Histol.,9(5):375-82] describes the use of a time-delay and integration (TDI)detector in an imaging flow cytometer. A TDI detector is any pixilateddevice in which the signal produced in response to radiation directed atthe device can be caused to move in a controlled fashion. Typically, thepixels of a TDI detector are arranged in rows and columns, and thesignal is moved from row to row in synchrony with a moving imageprojected onto the device, allowing an extended integration time withoutblurring. The approach disclosed by Ong et al. advanced the art byaddressing the need for spatial resolution and high sensitivity forcells in flow. However, this approach does not address the remainingprincipal characteristics. The authors of this paper cite an operatingspeed of 10 cells per second and a theoretical speed limitation of 500cells per second, which is at least an order of magnitude slower than isrequired for non-invasive fetal testing. In addition, the system has nospectral resolution; laser scatter and fluorescent light are collectedby the imaging system indiscriminately.

In more recent developments, U.S. Pat. No. 5,644,388 discloses analternative approach to an imaging flow cytometer. The patent disclosesthe use of a frame-based image collection approach in which a videocamera views cells in flow, in a freeze frame fashion. This methodrequires the image collection system to be synchronized with thepresence of cells in the imaging area, unlike the case of TDI, whereinthe detector readout rate is synchronized with the velocity of thecells. When a cell is imaged with the frame-based method, theintegration period must be very short to prevent blurring. A shortintegration time is achieved either with a strobed light source, or acontinuous light source combined with a shuttered detector. In eithercase, the short integration time reduces the signal-to-noise ratio andthe ultimate sensitivity of the approach. Further, frame-based camerasrequire time to transfer data out of the camera, during which no imagesare acquired, and cells of interest can escape detection. Finally, likethe work of Ong et al., this patent makes no provisions for acquiringdata over a large spectral bandwidth and with sufficient spectralresolution to simultaneously resolve numerous and differently coloredfluorescent probes and FISH spots.

Spectral discrimination is addressed in U.S. Pat. No. 5,422,712, inwhich the spectra of particles suspended in a fluid are collected as theparticles flow through a detection region. However, there is no spatialrepresentation of the object in the system disclosed in this patent,because the object is defocussed at the detector. In this system, lightis collected from the object and an image is created at an intermediateaperture. The light continues through the aperture to a spectraldispersing element, which disperses the light spectrally along the axisof flow. The dispersed light is applied to an image intensifier in whichit is amplified, and the light signal output from the image intensifieris finally directed to frame-based detector. At the intermediateaperture, prior to spectral dispersion, the image represents the spatialdistribution of light in object space. The spatial distribution isblurred as the light propagates past the image plane, through thespectral dispersing element and onto the image intensifier. Becausethere is no provision for re-imaging the intermediate aperture at theintensifier, the resulting signal distribution at the intensifierrepresents only the spectral distribution of the light and does notpreserve the spatial distribution of the light from the object. The lossof spatial information limits the utility of the invention forapplications such as fetal cell analysis. If multiple identical FISHspots are present in a cell, their spectra can be ascertained using thisapproach, but the number of spots cannot be determined. In addition,this approach disperses the wavelength spectrum parallel to the axis offlow. If two particles are illuminated in the flow axis, their spectracan overlap on the detector. To prevent this problem, the patentdiscloses that a very short illumination height in the flow axis isused. The short illumination height decreases integration time, whichnecessitates the use of the image intensifier. Further, the shortillumination height limits throughput by preventing the simultaneousimaging of multiple cells in the flow axis.

Accordingly, it will be apparent that an improved technique is desiredthat resolves the limitations of the conventional approaches discussedabove. It is expected that the new approach developed to address theseproblems in the prior art will also have application to the analysis ofother types of moving objects besides cells and may be implemented indifferent configurations to meet the specific requirements of disparateapplications of the technology.

SUMMARY OF THE INVENTION

The present invention is directed to an imaging system that is adaptedto determine one or more characteristics of an object from an image ofthe object. There is relative movement between the object and theimaging system, and although it is contemplated that either (or both)may be in motion, the object will preferably move while the imagingsystem will be fixed. In addition, it should also be understood thatwhile much of the following summary and the corresponding claims recite“an object,” it is clearly contemplated that the present invention ispreferably intended to be used with a plurality of objects and isparticularly useful in connection with imaging a stream of objects.

The present invention provides a method and apparatus for the analysisof rare cells in the blood for the purposes of non-invasive fetal celldiagnosis and cancer screening, as well as other applications. Toachieve such functions, the present invention is capable of rapidlycollecting data from a large cell population with high sensitivity andlow measurement variation. These data include simultaneous spatial andspectral images covering a large bandwidth at high resolution. Further,the present invention preserves the spatial origin of the spectralinformation gathered from the object.

Several different embodiments of the imaging system are provided. Onepreferred form of the invention includes a collection lens disposed sothat light traveling from the object is collimated by passing throughthe collection lens and travels along a collection path. A spectraldispersing element is disposed in the collection path so as tospectrally disperse the collimated light that has passed through thecollection lens in a plane substantially orthogonal to a direction ofrelative movement between the object and the imaging system, producingspectrally dispersed light. (As noted above, the object or the imagingsystem or both can be in motion relative to the other and for the sakeof simplicity, this relative movement is hereinafter referred to simplyas “the movement.”) An imaging lens is disposed to receive thespectrally dispersed light, producing an image from the spectrallydispersed light. Also included is a TDI detector disposed to receive theimage produced by the imaging lens. As the movement occurs, the image ofthe object produced by the imaging lens moves from row to row across theTDI detector. The TDI detector produces an output signal that isindicative of at least one characteristic of the object, by integratinglight from at least a portion of the object over time.

As a result of light collimation by the collection lens in thisembodiment, all light emitted from a first point in the object travelsin parallel rays. Light emitted from a second point in the object willalso travel in parallel rays, but at a different angle relative to lightfrom the first point. In this manner, spatial information in the objectis transformed by the collection lens into angular information in thecollection path. The spectral dispersing element acts on the collimatedlight such that different spectral components leave the spectraldispersing element at different angles, in a plane substantiallyorthogonal to the direction of the movement between the object and theimaging system. In this manner, both spatial and spectral information inthe object are transformed into angular information. The imaging lensacts on the light from the dispersing element to transform differentlight angles into different positions on the detector. Spatialinformation is preserved by the system since light from the differentpositions in the object is projected to different positions on thedetector, for both axes. In addition, light of different spectralcomposition that originates from the object is projected to differentpositions on the detector in an axis substantially orthogonal to themovement. In this manner, the spatial information from the object ispreserved, while spectral information covering a large bandwidth issimultaneously collected at high resolution.

FIG. 16 further illustrates the simultaneous collection of spectral andspatial information by the present invention, when imaging male andfemale cells 200 and 208, respectively. Light of shorter wavelength,such as green laser scatter 212, will be focussed on the left side ofthe TDI detector. Light of slightly longer wavelength, such as yellowfluorescence 214 from a cell nucleus 202 or 210, will be laterallyoffset to the right. Light of still longer wavelengths, such as orangefluorescence 216 from an X-chromosome FISH probe and red fluorescence218 from a Y-chromosome FISH probe, will be focussed progressivelyfarther to the right on the TDI detector. In this manner, differentcomponents of a cell that fluoresce at different wavelengths will befocussed at different locations on the TDI detector, while preservingthe spatial information of those components. Each component image may bebroadened laterally due to the width of its associated fluorescenceemission spectrum. However, this broadening can be corrected based upona priori knowledge of the emission spectra. Deconvolution of theemission spectrum from the broadened component image will yield anundistorted component image. Further, since the spectral dispersioncharacteristics of the spectral dispersing element are known, thelateral offsets of the different color component images can be correctedto reconstruct an accurate image of the cell. Using this embodiment ofthe invention, high spatial resolution information can be collectedsimultaneously with high spectral resolution over several hundrednanometers of spectral bandwidth. It should clear to those skilled inthe art that the present invention can be employed to enumerate numerousand multicolored FISH probes to simultaneously determine manycharacteristics from cells.

In the following disclosure, for all forms of the present inventionwhere a prism is used as a spectral dispersing element, it can bereplaced with a spectral dispersing component having characteristicsthat ensure no distortion or convolution of the image occurs due to theemission bandwidth, and as a result, a deconvolution is not needed tocorrect the image. Preferably, the spectral dispersing componentemployed comprises a plurality of dichroic beam splitters, such asdichroic mirrors, which are arranged to reflect light within predefinedbandwidths at predefined angles. Unlike a prism, where every wavelengthleaves the prism at a different angle, all light within a predefinedbandwidth incident on the dichroic beam splitter at a common angleleaves a given dichroic beam splitter at the same angle. Therefore,there is no convolution between the emission spectrum of the lightleaving the object and the image of that object. When using such aspectral dispersing component, light of a first spectral bandwidthreflects off the first dichroic beam splitter at a predefined nominalangle. Light of a second spectral bandwidth is passed through the firstdichroic beam splitter to the next dichroic beam splitter and isreflected therefrom at a different predefined nominal angle. Light of athird spectral bandwidth is passed through the first and second dichroicbeam splitters to a third dichroic beam splitter and reflected therefromat a third predefined nominal angle. The dichroic beam splitters areselected to cover the desired light spectrum with the appropriatespectral passbands. The angle of each dichroic beam splitter is set suchthat light reflected from it within the corresponding spectral bandwidthfor the dichroic beam splitter is focussed onto a different region ofthe detector. Since the present invention uses a narrow field angle inobject space along the axis perpendicular to the axis of motion, manydifferent spectral bandwidths can be simultaneously imaged onto a singledetector. In this manner, each region on the detector may cover adifferent spectral bandwidth while collecting light over the same fieldangle in object space.

Depending on the amount of out-of-band rejection required, a bandpassfilter is optionally placed in front of the detector. In one embodiment,the bandpass filter comprises a plurality of narrow spectral filtersplaced side-by-side to cover regions of the detector in correspondencewith the spectral information to be imaged in those regions. Since theposition of each spectral bandwidth region is predefined, and since thepresent invention maintains the spatial integrity of the object, a fullcolor, high spectral resolution representation of the object isgenerated from the spectral information imaged onto the detector.

The use of a TDI detector in the present invention results in anextended imaging region along the axis of motion and a correspondinglylong integration time. Several light sources can be simultaneouslyprojected into the imaging region, increasing the amount of lightincident upon objects therein. In addition, the combination of anextended imaging region and the orthogonal orientation of the spectraldispersion axis relative to the axis of the motion allows multipleobjects to be imaged simultaneously. The long integration time andparallel image acquisition of this embodiment allows sensitive andconsistent imaging performance to be combined with high throughput.

There are several alternative ways to provide light from the object. Inone case, the light from the object comprises an unstimulated emissionfrom the object, i.e., the object emits light without requiring a lightsource to stimulate the emission. In another embodiment, a light sourceis disposed to provide an incident light that illuminates the object. Inthis case, the object may scatter the incident light so that the lightscattered from the object at least in part passes through the collectionlens, or the incident light illuminating the object may stimulate theobject to emit the light that passes through the collection lens.Further, the incident light may at least be partially absorbed by theobject, so that the light passing through the collection lens does notinclude a portion of the light absorbed by the object. Finally, theincident light from the light source may be reflected from the objecttoward the collection lens. The light source or sources that are usedpreferably comprise at least one of a coherent light source, anon-coherent light source, a pulsed light source, and a continuous lightsource.

Spectral dispersion may be accomplished by many means, including a prismor grating. Further, although one preferred form of the inventionemploys a spectral dispersing element, the present invention is notlimited to imaging the spectral dispersion of light. Alternatively, adispersing element can be used to disperse light as a function ofposition, angle, polarization, phase, and other properties.

The object may be entrained within a fluid stream that moves the objectpast the collection lens, or alternatively, can be carried on a support,or simply move without the benefit of a support or flowing medium.Moreover, the present invention is not limited to the imaging ofmicroscopic or small objects.

The TDI detector preferably responds to the image of the object byproducing a signal that propagates across the TDI detector. Pixels of atypical TDI detector are arranged in rows and columns, and the signalpropagates from row to row. However, the present invention is notlimited to TDI detectors employing a rectilinear arrangement of pixels(e.g., a microchannel plate-based TDI detector). A propagation rate ofthe signal across the TDI detector can either be synchronized with amotion of the image of the object on the TDI detector as a result of themovement, or can be non-synchronized with the movement.

Other aspects of the present invention are directed to methods forimaging an object. These methods implement steps that are generallyconsistent with the imaging system discussed above.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same becomesbetter understood by reference to the following detailed description,when taken in conjunction with the accompanying drawings, wherein:

FIG. 1 is a plan view of a first embodiment of the present invention inwhich particles conveyed by a fluid stream depicted as flowing into thesheet;

FIG. 2 is a side elevational view of the first embodiment shown in FIG.1;

FIG. 3 is an isometric view of the first embodiment of FIG. 1;

FIG. 4 is an isometric view of a confocal embodiment that includes aslit that is used for spatial filtering of extraneous light;

FIG. 5 is an isometric view showing different locations for a lightsource in connection with the first embodiment;

FIG. 6 is an alternative to the first embodiment in which a second setof imaging components and TDI detector is included for monitoring lightfrom a particle, to avoid interference between FISH probes, and showingalternative locations for light sources;

FIG. 7 is an isometric view of an embodiment in which an object issupported by or comprises a slide that moves past a collection lens,showing different locations for a light source;

FIGS. 8A and 8B are respectively a plan view and a side elevational viewof an alternative to the embodiment of FIG. 7 that is used to produce ascattered pattern on the TDI detector;

FIG. 9 is a plan view of yet a further embodiment in which light forminga scatter patterned image and spectrally dispersed light from the objectare imaged on separate portions of a TDI detector;

FIG. 10 is a plan view of a still further embodiment in which lightforming a scatter patterned image and spectrally dispersed light fromthe object are imaged by two different TDI detectors;

FIG. 11 is a schematic diagram illustrating the optical convolution of anarrow FISH emission spectrum by the present invention, to resolve twoFISH probes in a cell;

FIG. 12 is a schematic diagram showing the optical convolution of twodifferent colors of narrow FISH emission spectra, to resolve the imageof the FISH probes on the TDI detector;

FIG. 13 is a schematic diagram illustrating how for a wider FISHemission spectrum, a deconvolution is provided by the present inventionto resolve the image of two FISH probes of a single color;

FIG. 14 is a schematic diagram showing the deconvolution of two colorFISH spectra that are relatively wide, to resolve the image of the FISHprobes;

FIG. 15 is a schematic block diagram of the system used to process thesignal produced by a TDI detector in the present invention;

FIG. 16 is a schematic diagram illustrating how the present invention isused to determine whether a cell is from a male or female;

FIG. 17 is a plan view of an alternate embodiment that employs aspectral dispersion component comprising a plurality of stacked dichroicfilters employed to spectrally separate the light;

FIG. 18 is an X-Y plot of several typical passbands for the dichroicfilters employed in the embodiment shown in FIG. 17;

FIG. 19 is a schematic illustration of a detection filter assembly thatmay optionally be placed in front of the TDI detector in the embodimentof FIG. 17 to further suppress out-of-band light;

FIGS. 20A-20E are X-Y plots of transmission vs. wavelength correspondingto corresponding passbands of the filter segments of the detectionfilter assembly that may optionally be placed in front of the TDIdetector;

FIG. 21 is a plan view of another embodiment of the configuration ofFIG. 17, wherein the spectral dispersion filter system comprises aplurality of dichroic cube filters orientated at various angles tocreate the spectral dispersing effect; and

FIG. 22 illustrates an exemplary set of images projected onto the TDIdetector when using the spectral dispersing filter system of the FIG.17.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention offers considerable advantages over systemsemployed for cell and particle analysis in the prior art. Theseadvantages arise from the use in the present invention of an opticaldispersion system in combination with a TDI detector that produces anoutput signal in response to the images of cells and other objects thatare directed on the TDI detector. Multiple objects can be imaged on theTDI detector at the same time. In addition, the image of each object canbe spectrally decomposed to discriminate object features by absorption,scatter, reflection or probe emissions using a common TDI detector foranalysis.

The present invention can be employed to determine morphological,photometric, and spectral characteristics of cells and other objects bymeasuring optical signals including light scatter, reflection,absorption, fluorescence, phosphorescence, luminescence, etc.Morphological parameters include nuclear area, perimeter, texture orspatial frequency content, centroid position, shape (i.e., round,elliptical, barbell-shaped, etc.), volume, and ratios of any of theseparameters. Similar parameters can also be determined for the cytoplasmof cells with the present invention. Photometric measurements with theinvention enable the determination of nuclear optical density, cytoplasmoptical density, background optical density, and the ratios of any ofthese values. An object being imaged with the present invention caneither be stimulated into fluorescence or phosphorescence to emit light,or may be luminescent, producing light without stimulation. In eachcase, the light from the object is imaged on the TDI detector of thepresent invention to determine the presence and amplitude of the emittedlight, the number of discrete positions in a cell or other object fromwhich the light signal(s) originate(s), the relative placement of thesignal sources, and the color (wavelength or waveband) of the lightemitted at each position in the object.

An initial application of the imaging system comprising the presentinvention will likely be employed as a cell analyzer to determine one ormore of the parameters listed above, for cells entrained in a fluidflowing through the imaging system. However, it should also beunderstood that this invention can be used for imaging other movingobjects.

First Preferred Embodiment

A first preferred embodiment of an imaging system 20 in accord with thepresent invention is schematically illustrated in FIGS. 1, 2, and 3, inconnection with producing images of moving objects such as cells thatare conveyed by a fluid flow 22 through the imaging system. In FIG. 1,fluid flow 22 entrains an object 24 (such as a cell, but alternatively,a small particle) and carries the object through the imaging system. Thedirection of the fluid flow in FIG. 1 is into (or out of) the sheet,while in FIGS. 2 and 3, the direction of flow is from top to bottom, asindicated by the arrow to the left of the Figures. Light 30 from object24 passes through collection lenses 32 a and 32 b that collect thelight, producing collected light 34, which is approximately focussed atinfinity, i.e. the rays of collected light from collection lens 32 b aregenerally parallel. Collected light 34 enters a prism 36, whichdisperses the light, producing dispersed light 38. The dispersed lightthen enters imaging lenses 40 a and 40 b, which focuses light 42 onto aTDI detector 44.

As will be evident in FIG. 2, if the Figure depicts the imaging ofobject 24 over time, the object is shown at both a position 26 and aposition 28 as it moves with fluid flow 22. As a consequence, images ofobject 24 will be produced on the detector at two discrete spatialpositions 26′ and 28′, as indicated on the right side of FIG. 2.Alternatively, if FIG. 2 is depicting a single instant in time,positions 26 and 28 can represent the location of two separate objects,which are simultaneously imaged on the detector at positions 26′ and28′.

In regard to imaging system 20 and all other imaging systems illustratedherein, it will be understood that the lenses and other optical elementsillustrated are shown only in a relatively simple form. Thus, thecollection lens is illustrated as a compound lens comprising onlycollection lenses 32 a and 32 b. Lens elements of different designs,either simpler or more complex, could be used in constructing theimaging system to provide the desired optical performance, as will beunderstood by those of ordinary skill in the art. The actual lenses oroptical elements used in the imaging system will depend upon theparticular type of imaging application for which the imaging system willbe employed.

In each of the embodiments of the present invention, it will beunderstood that relative movement exists between the object being imagedand the imaging system. In most cases, it will be more convenient tomove the object than to move the imaging system. However, it is alsocontemplated that in some cases, the object may remain stationary andthe imaging system move relative to it. As a further alternative, boththe imaging system and the object may be in motion but either indifferent directions or at different rates.

The TDI detector that is used in the various embodiments of the presentinvention preferably comprises a rectangular charge-coupled device (CCD)that employs a specialized pixel read out algorithm, as explained below.Non-TDI CCD arrays are commonly used for 2-dimensional imaging incameras. In a standard CCD array, photons that are incident on a pixelproduce charges that are trapped in the pixel. The photon charges fromeach pixel are read out of the detector array by shifting the chargesfrom one pixel to the next, and then onto an output capacitor, producinga voltage proportional to the charge. Between pixel readings, thecapacitor is discharged and the process is repeated for every pixel onthe chip. During the readout, the array must be shielded from any lightexposure to prevent charge generation in the pixels that have not yetbeen read.

In one type of TDI detector 44, which comprises a CCD array, the CCDarray remains exposed to the light as the pixels are read out. Thereadout occurs one row at a time from the top toward the bottom of thearray. Once a first row is read out, the remaining rows are shifted byone pixel in the direction of the row that has just been read. If theobject being imaged onto the array moves in synchrony with the motion ofthe pixels, light from the object is integrated for the duration of theTDI detector's total readout period without image blurring. The signalstrength produced by a TDI detector will increase linearly with theintegration period, which is proportional to the number of TDI rows, butthe noise will increase only as the square root of the integrationperiod, resulting in an overall increase in the signal-to-noise ratio bythe square root of the number of rows. One TDI detector suitable for usein the present invention is a Dalsa Corp., Type IL-E2 image sensor,although other equivalent or better image sensors can alternatively beused. The Dalsa image sensor has 96 stages or rows, each comprising 512pixels; other types of image sensors useable in the present inventionmay have different configurations of rows and columns or anon-rectilinear arrangement of pixels. The Dalsa sensor hasapproximately 96 times the sensitivity and nearly 10 times thesignal-to-noise ratio of a standard CCD array. The extended integrationtime associated with TDI detection also serves to average out temporaland spatial illumination variations, increasing measurement consistency.

In imaging system 20 and in other embodiments of the present inventionthat employ a fluid flow to carry objects through the imaging system, aflow-through cuvette or a jet (not shown) contains the cells or otherobjects being analyzed. The velocity and cellular concentration of thefluid may be controlled using syringe pumps, gas pressure, or otherpumping methods (not shown) to drive a sample solution through thesystem to match the pixel readout rate of the TDI detector. However, itshould be understood that the readout rate of the TDI detector can beselectively controlled, as required, to match the motion of the samplesolution.

Various optical magnifications can be used to achieve a desiredresolution of the object that is being imaged on the light sensitiveregions (pixels) of the TDI detector. It is contemplated that in mostembodiments, the optical magnification will fall within a range of 1:1to 50:1, providing a substantial range in the number of light sensitiveregions on the TDI detector on which images of the object are formed,also depending of course, on the actual size of the object being imagedand its distance from the imaging system. It is envisioned that thepresent invention can have applications ranging from the analysis ofcells and other microscopic objects to the imaging of stellar objects.

It should be emphasized that the present invention is not limited to CCDtypes of TDI detectors. Other types of TDI detectors, such ascomplementary metal oxide semiconductor (CMOS) and multi-channel plateimaging devices might also be used for the TDI detector in the presentinvention. It is important to understand that any pixellated device(i.e., having a multitude of light sensitive regions) in which a signalproduced in response to radiation directed at the device can be causedto move through the device in a controlled fashion is suitable for useas the TDI detector in the present invention. Typically, the signal willmove in synchrony with a moving image projected onto the device, therebyincreasing the integration time for the image, without causing blurring.However, the motion of the signal can be selectively desynchronized fromthe motion of the radiation image, as required to achieve a desiredaffect.

Second Preferred Embodiment

FIG. 4 illustrates an imaging system 45, which is a second preferredembodiment of the present invention and which is similar in many ways toimaging system 20. However, imaging system 45 is a confocal embodimentthat includes a slit 52 that substantially prevents extraneous lightfrom reaching TDI detector 44. In imaging system 45, light 46 fromobject 24 is focussed by an objective lens 48 onto a slit 52. Slit 52,as shown in FIG. 4, is sufficiently narrow to block light which is notfocussed onto the slit by objective lens 48 from passing through theslit. Light 30′ passes through the slit and is collected by collectionlens 32 as discussed above, in regard to imaging system 20. Collectedlight 34 is spectrally dispersed by prism 36, and is imaged by imaginglens 40 onto TDI detector 44, also as discussed above. By excludinglight other than that from object 24 from reaching TDI detector 44, theTDI detector produces an output signal that corresponds only to theactual images of the object, and the signal is not affected by theextraneous light, which has been excluded. If not excluded in thismanner, the ambient light reaching TDI detector 44 might otherwiseproduce “noise” in the output signal from the TDI detector.

It should be noted that in the illustration of each of imaging systems20 and 45, a light source has not been shown. These first twoembodiments have been illustrated in their most general form to makeclear that a separate light source is not required to produce an imageof the object, if the object is luminescent, i.e., if the objectproduces light. However, many of the applications of the presentinvention will require that one or more light sources be used to providelight that is incident on the object being imaged. The location of thelight sources substantially affects the interaction of the incidentlight with the object and the kind of information that can be obtainedfrom the images on the TDI detector.

In FIG. 5, several different locations of light sources usable toprovide light incident on object 24 are illustrated. It should beunderstood, however, that light sources can be located at many otherpositions besides those shown in FIG. 5. The location of each one ormore light source employed will be dependent upon the kind of imaging ofthe object, and the kind of data for the object, to be derived from thesignal produced by the TDI detector. For example, employing a lightsource 60 a or a light source 60 b, as shown in the Figure, will providelight 58 that is incident on object 24 and which is scattered from theobject into the optical axis of collection lens 32. The optical axis ofcollection lens 32 is at about a 90° angle relative to the directions ofthe light incident upon object 24 from either light source 60 a or 60 b.

In contrast, a light source 62 is disposed so that light 58 emitted fromthe source travels toward the object in a direction that is generallyaligned with the optical axis of collection lens 32, so that the imageformed on TDI detector 44 will not include light absorbed by object 24.Light absorption characteristics of the object can thus be determined byilluminating the object using a light source 62.

A light source 64 is disposed to illuminate object 24 with lightdirected toward the object along a path that is approximately 30-45° offthe optical axis of collection lens 32. This light 58, when incident onobject 24 will be reflected (scattered) from object 24, and thereflected or scattered light will be imaged on TDI detector 44. A moredirectly reflected light is provided by an epi light source 66, disposedso as to direct its light 58 toward a partially reflective surface 68that is disposed so that a portion of the light is reflected throughcollection lens 32 and onto object 24. The light reaching the objectwill be reflected from it back along the axis of collection lens 32 andwill at least in part pass through partially reflective surface 68 toform an image of the object on TDI detector 44. Alternatively, adichroic mirror may be employed instead of, and in the position of,partially reflective surface 68 to direct light from epi light source 66to excite fluorescence or other stimulated emission from object 24.Emission from object 24 is then at least partially collected bycollection lens 32 and passes through the dichroic mirror for spectraldispersion and detection by the TDI detector.

In addition to imaging an object with the light that is incident on it,a light source can also be used to stimulate emission of light from theobject. For example, FISH probes that have been inserted into cells willfluoresce when excited by light, producing a correspondingcharacteristic emission spectra from any excited FISH probe that can beimaged on TDI detector 44. In FIG. 5, light sources 60 a, 60 b, 64, or66 could alternatively be used for causing the excitation of FISH probeson object 24, enabling TDI detector 44 to image FISH spots produced bythe FISH probes on the TDI detector at different locations as a resultof the spectral dispersion of the light from the object that is providedby prism 36. The disposition of these FISH spots on the TDI detectorsurface will depend upon their emission spectra and their location inthe object. Use of FISH probes in connection with producing images ofFISH spots on the TDI detector with the present invention is discussedin greater detail below.

Each of the light sources illustrated in FIG. 5 produces light 58, whichcan either be coherent, non-coherent, broadband or narrowband light,depending upon the application of the imaging system desired. Thus, atungsten filament light source can be used for applications in which anarrowband light source is not required. For applications such asstimulating the emission of fluorescence from FISH probes, narrowbandlaser light is preferred, since it also enables a spectrally-decomposed,non-distorted image of the object to be produced from light scattered bythe object. This scattered light image will be separately resolved fromthe FISH spots produced on TDI detector 44, so long as the emissionspectra of any FISH spots are at different wavelengths than thewavelength of the laser light. The light source can be either of thecontinuous wave (CW) or pulsed type. If a pulsed type illuminationsource is employed, the extended integration period associated with TDIdetection can allow the integration of signal from multiple pulses.Furthermore, it is not necessary for the light to be pulsed insynchronization with the TDI detector.

Pulsed lasers offer several advantages over CW lasers as a light sourcein the present invention, including smaller size, higher efficiency,higher reliability, and the ability to deliver numerous wavelengthssimultaneously. Another advantage of pulsed lasers is their ability toachieve saturating levels of fluorescence excitation of fluorescentprobes used in cells. Fluorescence saturation occurs when the number ofphotons encountering a fluorescent molecule exceeds its absorptioncapacity. Saturating excitation produced by a pulsed laser is inherentlyless noisy than unsaturating CW laser excitation because variations inpulse-to-pulse excitation intensity have little effect on thefluorescence emission intensity.

Prism 36 in the imaging systems discussed above can be replaced with adiffraction grating, since either is capable of spectrally dispersingthe optical signals from the cells over the pixels of the TDI detector.In addition to providing useful data from a cell or other object,spectral dispersion can be used to reduce measurement noise. In caseswhere the light source wavelength differs from the emission spectra ofthe fluorescent probes, the light from the source that is scattered intothe collection system is spatially isolated from the fluorescencesignals. If the light source wavelength overlaps the emission spectra ofthe fluorescent probes, the pixels of the TDI detector in which light ofthe wavelength of the source falls can be isolated from those pixels onwhich the remaining fluorescence signals fall. Further, by dispersingthe fluorescence signals over multiple pixels, the overall dynamic rangeof the imaging system is increased.

Third Preferred Embodiment

A third preferred embodiment is a stereoscopic arrangement 70 of thefirst preferred embodiment, as illustrated in FIG. 6. This arrangementallows the imaging of the object from two different directions in orderto distinguish features that would otherwise overlap when viewed from asingle direction. While the third preferred embodiment can be employedfor objects on moving substrates such as microscope slides, it isparticularly useful for analyzing multi-component objects in solution,such as cells containing FISH probes. Such probes appear as pointsources of light anywhere within the cell's three dimensional nucleus.In some cases, two or more FISH probes may appear in an overlappingrelationship along the optical axis of the imaging system. In suchcases, one of the FISH probes may obscure the others, making itdifficult to determine the number of probes present in the cell. This isa key factor in the determination of genetic abnormalities such astrisomy 21, otherwise known as Down syndrome. Single-perspective systemsmay address this problem by “panning through” the object along theoptical axis to acquire multiple image planes in the object. While thismethod may be effective, it requires a significant amount of time tocollect multiple images and cannot be readily applied to a cell in flow.The stereoscopic imaging system 70 in FIG. 6 includes two TDI detectors44 a and 44 b, and their associated optical components, as discussedabove in connection with imaging system 20.

By positioning the optical axes of collection lenses 32 for the two TDIdetectors so that they are spaced apart, for example, by 90°, it ispossible to separately resolve the FISH spots imaged from two or moreFISH probes on at least one of TDI detectors 44 a or 44 b. If two ormore FISH probes overlap in regard to the image produced on one of thedetectors, they will be separately resolved in the spectrally dispersedimages produced on the other TDI detector. Further, the use of two TDIdetectors in imaging system 70 in what might be referred to as a “stereoor three-dimensional configuration” allows flexibility in theconfiguration of each leg of the system, including parameters such asthe relative TDI readout rates, axial orientations, inclinations, focalplane positions and magnification. Multiple cells or other objects maybe imaged onto each detector simultaneously in the vertical direction.Since the objects may move in synchronicity with the signal on the TDI,no gate or shutter is required to prevent blurring of the image. Aspreviously noted, the present invention can use a pulsed or CW lightsource without need for a trigger mechanism to time a pulse coincidentwith particle arrival in the field of view. If a pulsed light source isused, the extended field of view in the axis of motion associated withTDI detection allows the cell or object in motion to be illuminated bymultiple pulses during its traversal. In contrast to a frame-basedimaging apparatus, a TDI system can produce a single unblurred image ofthe object that integrates the signal from multiple pulses. When a CWlight source is used, the signal generated by the object will becollected throughout the entire traversal of the object through thefield of view, as opposed to only a small segment in time when a shutteris open. Therefore, the amount of signal collected and imaged on thedetector in the present invention is substantially greater than that ofthe prior art frame-based imaging systems. Consequently, the presentinvention can operate at very high throughput rates with excellentsignal-to-noise ratio.

Also illustrated in FIG. 6 are several exemplary positions for lightsources, which are useful for different purposes in connection with theimaging system illustrated therein. In connection with TDI detector 44a, light source 62 provides illumination of object 24 from a directionso that absorption characteristics of the object can be determined fromthe image produced on the TDI detector. At the same time, light providedby light source 62 that is scattered from object 24 can be used toproduce a scatter image and spectrally dispersed images on TDI detector44 b. Light source 74 can be employed to produce spectrally dispersedand scattered images on both TDI detectors 44 a and 44 b. If lightsources 62 and 72 are of different wavelengths and an appropriate filteris provided to block the wavelength from the light source aligned withthe optical axis of the respective collections lenses 32, these twolight sources can be used for producing scattered light from the object.For example, suppose light source 72 produces light of a wavelength Athat scatters from object 24 and is directed toward TDI detector 44 a.By including a filter (not shown) that blocks wavelength B produced bylight source 62, the light at wavelength B will not directly affect theimages produced on TDI detector 44 a. Similarly, the light from lightsource 72 would be blocked with an appropriate filter (not shown) sothat it does not interfere with the imaging of light produced by lightsource 62 that is scattered from object 24 onto TDI detector 44 b.

Epi light source 66 is also illustrated for use in producing images onTDI detector 44 a in conjunction with partial reflector 68. Light source64 can be used to generate reflected light to produce images on TDIdetector 44 a, while scattered light from this source is directed towardTDI detector 44 b. These and other possible locations of light sourceswill be apparent to those of ordinary skill in the art, as appropriatefor providing the incident light on the object needed to achieveimaging, depending upon the particular application and information aboutthe object that is desired.

Imaging Slide or Object Carried by Slide

Turning now to FIG. 7, an imaging system 80 is illustrated that issimilar to imaging system 20, except that it is used for imaging object24 on a slide 82. Object 24 is supported by slide 82 and the slide movesrelative to the imaging system as shown in FIG. 7. Alternatively, slide82 may be the object that is imaged. The object may be a semiconductorwafer, paper, or other object of interest since the object may be imagedusing reflected incident light.

To provide light incident on either slide 82 or object 24 that issupported thereby, a light source placed at one of several differentlocations can be employed. Exemplary light sources 62, 64, and 66illustrate some of the locations at which light sources useful in thisembodiment may be disposed. Light 58 emitted by any of the light sourcescan be either coherent or non-coherent light, pulsed or CW, and can bedirected through slide 82 (if it is transparent) from light source 62 orcan be reflected from the object or slide, if light sources 64 or 66 areemployed. As noted previously, epi light source 66 illuminates theobject in connection with a partially reflective surface 68.

Fourth Preferred Embodiment

FIGS. 8A and 8B show two different views of a fourth preferredembodiment, which is an imaging system 90 that produces a scatteredpattern image of object 24 on TDI detector 44. Light 30 from object 24passes through collection lenses 32 a and 32 b, and collected light 34is directed onto a cylindrical lens 92, as in the previous embodiments.Cylindrical lens 92 focuses light 94 on TDI detector 44, generally alonga line that is aligned with a central axis 96 of cylindrical lens 92.Central axis 96 is shown in FIG. 8B, and it will be apparent that it isorthogonal to the direction in which object 24 moves through the imagingsystem. As object 24 moves downwardly, relative to its disposition asshown in FIG. 8A, the focus of cylindrical lens 92 on TDI detector 44moves upwardly. Cylindrical lens 92 thus distributes an image of theobject along a row or rows of the light sensitive regions or pixels ofTDI detector 44.

Fifth Preferred Embodiment

Referring now to FIG. 9, an illustration of a fifth preferred embodimentis provided of an imaging system 100 that produces both a scatteredpattern image and a spectrally dispersed image of object 24 on TDIdetector 44. In imaging system 100, light 30 from object 24 passesthrough collections lenses 32 a and 32 b, which produce infinitelyfocussed light 34 directed toward a dichroic filter 102. Dichroic filter102 reflects light of a specific wavelength, e.g., the wavelength of alight source (not shown) that is incident upon object 24. Light of anyother wavelength is transmitted through dichroic filter 102 toward adiffraction grating 112. Diffraction grating 112 spectrally dispersesthe light transmitted through dichroic filter 102, which typically wouldbe light produced by the fluorescence of FISH probes on object 24, sothat a plurality of FISH spots corresponding to the number of differentFISH probes and objects being imaged are produced on TDI detector 44.

Light 104, which is reflected from dichroic filter 102 is transmittedinto cylindrical lens 106 and is focussed along a line as a scatteredpattern image in a region 110 on the TDI detector. The spectrallydispersed images of FISH spots or other aspects of object 24 havingwavelengths different than that reflected by dichroic filter 102 areimaged as light 116 by imaging lenses 114 a and 114 b onto a region 118of the TDI detector. Thus, signals corresponding to the scatteredpattern image and the spectrally dispersed images are both produced byTDI detector 44.

Sixth Preferred Embodiment

A sixth preferred embodiment, as illustrated in FIG. 10, is an imagingsystem 120 that is slightly different than the preceding fifthembodiment, since a dichroic filter 102′ is employed that is angled in adifferent direction, toward a second TDI detector 44 b. A dispersedpattern image represented by light 108′ is produced by a cylindricallens 106′ in this embodiment. Just as in imaging system 100, lighttransmitted through dichroic filter 102′ is focussed onto TDI detector44 a. Aside from using two separate TDI detectors that are disposed atdifferent sides of the imaging system, imaging system 120 issubstantially identical in operation to imaging system 100. However,just as in the third preferred embodiment, the use of two separate TDIdetectors allows flexibility in the configuration of each leg of thesystem, including parameters such as the relative TDI readout rates,axial orientations, inclinations, focal plane positions, andmagnification. It should also be noted that imaging system 100 could beconstructed to include two separate TDI detectors instead of a singleTDI detector, if desired.

Processing of Spectrally Dispersed Images on TDI Detector

When used for cell analysis, the present invention provides substantialutility in resolving FISH spots on the TDI detector, even when the FISHprobes are disposed in spatially close relationship within the cell.When spectral imaging occurs in the present invention, the spatialdistribution of light in the object is convolved with the spectraldistribution of that light to produce the image of the object at the TDIdetector. This convolution can result in blurring in the dispersionaxis, depending on the spectral bandwidth of the light. Narrow spectralbandwidths will result in little or no blurring depending on thespectral resolution of the system. In the present invention, it iscontemplated that the spectral resolution will be approximately 3 nm perpixel, with a spatial resolution in object space of approximately 1micron. However, the spatial and spectral resolution can be adjusted tomatch the requirements of the particular application.

FIG. 11 illustrates the present invention with a spectral resolution ofapproximately 10 nm per pixel and a spatial resolution of approximately0.5 microns. This Figure further illustrates how the present inventionis used to image a cell 140 having a nucleus 142 in which are disposedtwo FISH probes 144 a and 144 b having the same emission spectrum. InFIG. 11, the emission spectrum 146 of the FISH probes 144 a and 144 b isapproximately 10 nm in width, such as would be produced by “quantumdots” or a narrow-band fluorescent dye. The optical convolution of thenarrow bandwidth spectrum results in minimal blurring of FISH spots 148a and 148 b, enabling them to be readily resolved on TDI detector 44.

In FIG. 12, a cell 150 is illustrated having a nucleus 152 in which aredisposed FISH probes 154 and 156 having different emission spectra. FISHprobes are designed so that different emission spectra correspond todifferent DNA sequences. Each of the emission spectra of FISH probes 154and 156 are relatively narrow, as indicated by wavebands 158 and 160,and therefore, as in FIG. 11, minimal blurring occurs in FISH spots 162and 164. Furthermore, the spectral dispersion of the present invention,which maps wavelength into lateral position on TDI detector 44, producesa relatively wide physical displacement of FISH spots 162 and 164,despite the close proximity of FISH probes 154 and 156 in the cell.Taken together, FIGS. 11 and 12 illustrate how the present inventiondiscriminates FISH probes of the same or different color, therebyenabling the simultaneous enumeration of numerous genetic traits. Thoseskilled in the art can appreciate that the present invention is wellsuited to the requirements of fetal cell analysis, where there may beten or more probes of different colors present in the cell at one time.Further, those skilled in the art will appreciate that the presentinvention is not limited to the analysis of fetal cells using FISHprobes.

FIGS. 13 and 14 illustrate that the present invention can also be usedwith light of wide spectral bandwidth. In this case an additional signalprocessing step is performed to correct for lateral blurring due to thewide emission spectra. In FIG. 13, a cell 140 having a nucleus 142 isshown, and FISH probes 170 a and 170 b having a common emission spectrumare disposed in the nucleus. FISH probes 170 a and 170 b arecharacterized by producing a relatively wide emission spectrum 172. Whenoptically convolved by the spectral dispersion provided by the presentinvention, FISH spots 174 a and 174 b are produced on TDI detector 44,but their images are laterally blurred across TDI detector 44, as aresult of their relatively wide emission spectrum. To more clearlyresolve the separation of FISH spots 174 a and 174 b, a deconvolution iscarried out on the signal produced by TDI detector 44, with the knownFISH emission spectrum, thereby producing accurate FISH spotrepresentations 178 a and 178 b on a display 176. The deconvolution stepenhances the ability to enumerate the number of FISH spots.

FIG. 14 illustrates a corresponding relationship between FISH probes 180and 182, which are disposed within a nucleus 152 of a cell 150. FISHprobes 180 and 182 are characterized by each producing relatively wideband emission spectra 184 and 186, as shown in the Figure. Opticalconvolution of the fluorescence emitted by the FISH probes, which arespectrally dispersed, produces FISH spots 188 and 190 on TDI detector44. Again, by deconvolving the known FISH emission spectra with thesignal produced by TDI detector 44, the corresponding images shown ondisplay 176 of FISH spots 192 and 194 are recovered. Again, the spectraldispersion of the present invention, which maps wavelength into lateralposition on TDI detector 44, produces a relatively wide physicaldisplacement of FISH spots 192 and 194, despite the close proximity ofFISH probes 180 and 182 in the cell. In this manner, it is possible toresolve these images of FISH spots produced by FISH probes havingdifferent and relatively wide emission spectra.

A system 230 for analyzing the signal produced by TDI detector 44 andperforming the deconvolution steps described above is illustrated inFIG. 15. In this Figure, the signal from TDI detector 44 is applied toan amplifier 232, which buffers the signal and amplifies it to achieve alevel required by an analog to digital (A-D) converter 234. This A-Dconverter converts the analog signal from amplifier 232 into a digitalsignal that is input into a TDI line buffer 236. TDI line buffer 236temporarily stores the digital signal until it can be processed by a CPU238. To carry out the deconvolution noted above, a spectral buffer 240is loaded with the known emission spectrum for each of the FISH probesbeing used so that their emission spectra can be deconvolved with thesignal stored in TDI line buffer 236. CPU 238 is a high speed processorprogrammed to carry out the deconvolution and other analysis procedures,enabling the identification of desired characteristics or parameters ofthe object being imaged. The output from CPU 238 is temporarily storedin an image line buffer 242 that enables the image to be displayed orotherwise recorded for later analysis.

FIG. 16 illustrates a practical application of the present invention foridentifying a male cell 200 and a female cell 208 and for producingtheir corresponding scatter images 212 and 220. Male cell 200 includes anucleus 202 that has been stained with a yellow fluorescent dye. Inaddition, a FISH probe 204 produces a fluorescent orange emission,indicating the presence of an X-chromosome in the nucleus, while a FISHprobe 206 produces red fluorescence emission, indicating the presence ofa Y-chromosome. Spectral decomposition of the fluorescence emissionsfrom male cell 200, when the cell is illuminated with light from a greenlaser, results in a series of images on TDI detector 44, separated as afunction of the wavelength of the light that is imaged. Laser light thatis incident on the cells has an extremely narrow waveband, and image 212of male cell 200 produced by laser scatter is only slightly convolutedby the spectral decomposition process. Green laser scatter image 212 ofcell 200 and its nucleus 202 appear on the left side of the TDIdetector, while a fluorescent spot 214 corresponding to the yellowfluorescence emitted by nucleus 202 appears in the next few columns onthe TDI detector. Furthermore, as a function of the differentwavelengths of the fluorescence emitted by FISH probes 204 and 206, FISHspots 216 and 218 appear at locations spaced apart on the detector, butslightly blurred across the columns of TDI detector 44 due to the widthsof their respective emission spectra. By analyzing the signals producedby the TDI detector, the FISH probes responsive to X and Y chromosomesare detected, enabling the user to determine that cell 200 is a malecell, since it includes both the X and Y chromosome. Similarly, femalecell 208, when spectrally decomposed, also includes the characteristicyellow fluorescence of nucleus 210, but unlike the male cell, includestwo FISH spots 216 corresponding to FISH probes 204, which indicates thepresence of two X-chromosomes. Because TDI detector 44 alsodistinguishes the spatial position of male cell 200 and female cell 208,the corresponding spectral decompositions for these cells are readilyseparately resolved as both cells pass through the imaging system in thedirection indicated by the arrow to the lower left of FIG. 16. Again, itshould be noted that a deconvolution can be applied to the signalproduced by TDI detector 44 to provide better resolution of thecorresponding FISH spots that are illustrated.

Non-Distorting Spectral Dispersion Systems

The present invention can be provided with a spectral dispersion filterassembly that does not convolve the image with the emission spectra ofthe light forming the image, thereby eliminating the need fordeconvolution of the emission spectra from the image. FIG. 17illustrates a seventh preferred embodiment of the inventioncorresponding to such a non-distorting spectral dispersion system 250that employs a five color stacked wedge spectral dispersing filterassembly 252. This seventh embodiment is substantially similar to theembodiment shown in FIGS. 1, 2, and 3, except that spectral dispersingprism element 36 (of FIGS. 1, 2 and 3) is replaced by spectraldispersing filter assembly 252. The spectral dispersing filter assemblysplits the light into a plurality of light beams having differentbandwidths. Each light beam thus produced is directed at a differentnominal angle so as to fall upon a different region of TDI detector 44.The nominal angular separation between each bandwidth produced by thespectral dispersing filter assembly 252 exceeds the field angle of theimaging system in object space thereby preventing overlap of the fieldimages of various bandwidths on the detector.

Spectral dispersing filter assembly 252 comprises a plurality of stackeddichroic wedge filters, including a red dichroic filter R, an orangedichroic filter O, a yellow dichroic filter Y, a green dichroic filterG, and a blue dichroic filter B. Red dichroic filter R is placed in thepath of collected light 34, oriented at an angle of approximately 44.0°relative to an optic axis 253 of collection lenses 32 a and 32 b. Lightof red wavelengths and above, i.e., >640 nm, is reflected from thesurface of red dichroic filter R at a nominal angle of 1°, measuredcounter-clockwise from a vertical optic axis 257. Example spectralreflectance characteristics R′ of red dichroic filter R are plotted inFIG. 18, along with example spectral reflectance characteristicscorresponding to the other dichroic filters used in spectral dispersingfilter assembly 252. In FIG. 18, O′ indicates the spectral reflectancecharacteristics of orange dichroic filter O, Y′ indicates the spectralreflectance characteristics of yellow dichroic filter Y, etc. The lightreflected by red dichroic filter R leaves spectral dispersing filterassembly 252 and passes through imaging lenses 40 a and 40 b, whichcause the light to be imaged onto a red light receiving region of TDIdetector 44, which is disposed toward the right end of the TDI detector,as shown in FIG. 17.

Orange dichroic filter O is disposed a short distance behind reddichroic filter R and is oriented at an angle of 44.5 degrees withrespect to optic axis 253. Light of orange wavelengths and greater,i.e., >610 nm, is reflected by orange dichroic filter O at a nominalangle of 0.5° with respect to vertical optic axis 257. Because theportion of collected light 34 comprising wavelengths longer than 640 nmwas already reflected by red dichroic filter R, the light reflected fromthe surface of orange dichroic filter O is effectively bandpassed in theorange colored region between 610 nm and 640 nm. This light travels at anominal angle of 0.5° from vertical optic axis 257, and is imaged byimaging lenses 40 a and 40 b so as to fall onto an orange lightreceiving region disposed toward the right hand side of TDI detector 44between a center region of the TDI detector and the red light receivingregion, again as shown in FIG. 17.

Yellow dichroic filter Y is disposed a short distance behind orangedichroic filter O and is oriented at an angle of 45° with respect tooptic axis 253. Light of yellow wavelengths, i.e., 560 nm and longer, isreflected from yellow dichroic filter Y at a nominal angle of 0.0° withrespect to vertical optic axis 257. Wavelengths of light reflected byyellow dichroic filter Y are effectively bandpassed in the yellow regionbetween 560 nm and 610 nm and are imaged by imaging lenses 40 a and 40 bnear vertical optic axis 257 so as to fall on a yellow light receivingregion toward the center of TDI detector 44.

In a manner similar to dichroic filters R, O, and Y, dichroic filters Gand B are configured and oriented so as to image green and blue lightwavebands onto respective green and blue light receiving regions of TDIdetector 44, which are disposed toward the left-hand side of the TDIdetector. By stacking the dichroic filters at different predefinedangles, spectral dispersing filter assembly 252 collectively works tofocus light within predefined wavebands of the light spectrum ontopredefined regions of TDI detector 44. Those of ordinary skill in theart will appreciate that the filters used in the spectral dispersingfilter assembly 252 may have spectral characteristics that differ fromthose described above and in FIG. 18. Further, the spectralcharacteristics may be arbitrary and not limited to dichroic in order toachieve the desired dispersion characteristics.

The wedge shape of the dichroic filters in the preceding discussionallows the filters to be placed in near contact, in contact or possiblycemented together to form the spectral dispersing filter assembly 252.The angle of the wedge shape fabricated into the substrate for thedichroic filter allows easy assembly of the spectral dispersing filterassembly 252, forming a monolithic structure in which the wedge-shapedsubstrate is sandwiched between adjacent dichroic filters. If thefilters are in contact with each other or cemented together, thecomposition of the materials that determine the spectral performance ofthe filter may be different from those which are not in contact. Thoseof ordinary skill in the art will appreciate that flat, non wedge-shapedsubstrates could be used to fabricate the spectral dispersing filterassembly 252. In this case another means such as mechanically mountingthe filters could be used to maintain the angular relationships betweenthe filters.

In addition to the foregoing configuration, non-distorting spectraldispersion system 250 may optionally include a detector filter assembly254 to further attenuate undesired signals in each of the light beams,depending upon the amount of rejection required for out-of-band signals.FIG. 19 illustrates the construction of an exemplary detector filter 254corresponding to the five color bands discussed above and includes ablue spectral region 256, a green spectral region 258, a yellow spectralregion 260, an orange spectral region 262, and a red spectral region264, all of which are disposed side-by-side, as shown in the Figure. Thecorresponding spectral characteristics of the blue, green, yellow,orange, and red spectral regions or wavebands are respectively shown inFIGS. 20A-20E. The detection filter assembly shown in FIG. 19 may beconstructed by cementing separate filters in side-by-side arrangement ona common substrate or by other means well known to those or ordinaryskill in the art. Additionally, the ordinary practitioner in the artwill understand that the filter may alternatively be placed at anintermediate image plane, instead of directly in front of TDI detector44.

In the embodiment shown in FIG. 17, light may pass through each dichroicfilter in the spectral dispersing filter assembly 252 twice beforeexiting the spectral dispersing filter assembly 252. This condition willfurther attenuate out-of-band signals, but will also attenuate in-bandsignals. FIG. 21 illustrates an eighth embodiment of the presentinvention in which the light does not pass through another dichroicfilter after reflection. In this embodiment, a plurality of cubedichroic filters, including a red cube filter 266, a yellow cube filter268, a green cube filter 270, and a blue cube filter 272 are spacedapart sufficiently to ensure that light does not pass through any of thecube filters more than once. As with the embodiment of FIG. 17, the cubedichroic filters are oriented at appropriate angles to image lightwithin a predefined bandwidth to distinct regions on a TDI detector 274.As the light is reflected from each of cube dichroic filters 266, 268,270 and 272, it is directed toward imaging lenses 40 a and 40 b, anddifferent bandpass portions of the light are focussed upon correspondingred, yellow, green, and blue light receiving segments or regions definedon a light receiving surface of TDI detector 274. If desired, anoptional detector filter assembly 276 of similar construction todetector filter assembly 254 (but without the orange spectral region)may be used to increase the rejection of out-of-band signals. It shouldbe apparent to those skilled in the art that separate spaced apartplate, or pellical beam splitters could also be used in this applicationinstead of the cube filters. In the eight embodiment illustrated in FIG.21, the image lenses 40 a and 40 b must be placed a sufficient distanceaway from the plurality of cube filters to minimize the clear aperturerequirement for lenses 40 a and 40 b. Those skilled in the art willappreciate the clear aperture in the plane orthogonal to the page canincrease as the distance between the lenses and plurality cube filtersincreases. Therefore, the placement of lenses 40 a and 40 b must bechosen to appropriately accommodate the clear aperture in both planes.

The foregoing descriptions of the seventh and eighth preferredembodiments illustrate the use of four and five color systems. Thoseskilled in the art will appreciate that a spectral dispersing componentwith more or fewer filters may be used in these configurations in orderto construct a system covering a wider or a narrower spectral region, ordifferent passbands within a given spectral region. Likewise, thoseskilled in the art will appreciate that the spectral resolution of thepresent invention may be increased or decreased by appropriatelychoosing the number and spectral characteristics of the dichroic and orbandpass filters that are used. Furthermore, those skilled in the artwill appreciate that the angles or orientation of the filters may beadjusted to direct light of a given bandwidth onto any desired point onthe TDI detector. In addition, there is no need to focus the light inincreasing or decreasing order by wavelength. For example, influorescence imaging applications, one may wish to create more spatialseparation on the TDI detector between the excitation and emissionwavelengths by changing the angles at which the filters corresponding tothose wavelengths are oriented with respect to the optic axes of thesystem. Finally, it will be clear to those skilled in the art thatdispersion of the collected light may be performed on the basis ofnon-spectral characteristics, including angle, position, polarization,phase, or other optical properties.

As with the earlier embodiments discussed above, many applications ofthe seventh and eighth preferred embodiments will require that one ormore light sources be used to provide light that is incident on theobject being imaged. Accordingly, various light sources disposed atdifferent positions, such as those shown in FIGS. 5-7 and discussedabove, may be used to enhance the image quality produced by each ofthese embodiments. For clarity and to simplify the explanation of theseembodiments, the light sources have been omitted in FIGS. 17 and 21;however, it will be recognized by those skilled in the art how suchlight sources may be employed in these embodiments, based on theprevious discussion of the use of the light sources with respect to theearlier embodiments.

FIG. 22 illustrates the distribution of images on TDI detector 44corresponding to imaging a plurality of cells 200 when usingnon-distorting spectral dispersion system 250. As will be evident bycomparing FIG. 22 to FIG. 16, the resultant images on the TDI detectorare similar in many ways. However, when using the non-distortingspectral dispersion system, there is no image broadening as is seen inFIG. 22, which would otherwise result due to the convolution of theemission spectrum and the object. Instead, all wavelengths within thepredefined bandwidth of each dichroic filter are reflected from thefilter at the same nominal angle, so image components that fall withinthat passband suffer no positional distortion on the detector. The fieldangle orthogonal to flow in object space is also indicated on FIG. 22.In this particular configuration, the field angle in object space isless than +/−0.25°. Those skilled in the art will appreciate that thefield angle can be made larger or smaller. To the extent that the fieldangle is made larger, for example, to image cells over a wider region ona slide or in a broad flat flow, the field angle at the detector willincrease in proportion to the number of colors used. FIG. 22 illustratesthe image projected onto the detector when three cells 280, 282 and 284are flowing through the field of view. Light scatter images of cells280, 282, and 284 are seen on the left hand side of the detector denotedas the BLUE area. Images of cell nuclei 202 stained with a greenfluorescent dye are seen in the GREEN area of the detector. Threedifferently-colored genetic probes 204, 205, and 206 are also employedfor the analysis of the sex chromosomes within the cells. Probe 204stains the X chromosome with an orange fluorescing dye, probe 205 stainsthe Y chromosome with yellow fluorescing dye, and probe 206 stains theinactive X chromosome in female cells with a red fluorescing dye. Cell282 is imaged onto the detector as shown in FIG. 22. An image 286 ofprobe 204 from cell 282 is seen in the ORANGE area of the detector.Likewise an image 288 of probe 205 is seen in the YELLOW area of thedetector. The signal on the detector is processed to determine theexistence and position of these images on the detector to determine thatcell 282 is a male cell. In a similar manner, cells 280 and 284 containprobes 204 and 206, which create images 290 and 292 in the ORANGE areaof the detector, and images 294 and 296 in the RED area of the detector,indicating that these cells are female, respectively.

Although the present invention has been described in connection with thepreferred form of practicing it, those of ordinary skill in the art willunderstand that many modifications can be made thereto within the scopeof the claims that follow. Accordingly, it is not intended that thescope of the invention in any way be limited by the above description,but instead be determined entirely by reference to the claims thatfollow.

The invention in which an exclusive right is claimed is defined by thefollowing:
 1. An imaging system adapted to determine one or morecharacteristics of an object from an image of the object while there isrelative movement between the object and the imaging system comprising:(a) a collection lens disposed so that light traveling from the objectpasses through the collection lens and travels along a collection path;(b) a dispersing component disposed in the collection path so as toreceive the light that has passed through the collection lens,dispersing the light into a plurality of separate light beams, eachlight beam being directed away from the dispersing component in adifferent predetermined direction; (c) an imaging lens disposed toreceive the light beams from the dispersing component, producing aplurality of images corresponding to each of the light beams, each imagebeing projected by the imaging lens toward a different predeterminedlocation; and (d) a time delay integration (TDI) detector disposed toreceive the plurality of images produced by the imaging lens, producingan output signal that is indicative of at least one characteristic ofthe object, said TDI detector producing the output signal by integratinglight from at least a portion of the object over time, while therelative movement between the object and the imaging system occurs. 2.The imaging system of claim 1, wherein the dispersing componentcomprises a spectral dispersing component including a plurality of beamsplitters arranged to reflect light of predetermined spectralcharacteristics at different predefined angles, all light from each beamsplitter corresponding to a separate light beam, each light beam leavingthe spectral dispersing component at a different nominal angle.
 3. Theimaging system of claim 2, wherein the beam splitters are disposed inthe collection path adjacent to one another so as to receive the lightthat has passed through the collection lens, and arranged so that lightreflected by all but a first beam splitter in the spectral dispersingcomponent passes through at least one preceding beam splitters a secondtime.
 4. The imaging system of claim 3, wherein wedge-shaped substratesdefine an angular difference between each beam splitter, and wherein thebeam splitters are sandwiched between the wedge-shaped substrates,forming a monolithic structure.
 5. The imaging system of claim 2,wherein the beam splitters are disposed in the collection path so as toreceive the light that has passed through the collection lens andseparated from each other by a sufficient distance such that lightreflected by any of the beam splitters does not pass through any otherbeam splitter within the spectral dispersing component a second time. 6.The imaging system of claim 2, further including a bandpass filterassembly disposed between the TDI detector and the imaging lens, saidbandpass filter assembly comprising a plurality of adjacent filtersegments, each filter segment being positioned to receive a differentlight beam from an associated beam splitter in the spectral dispersingcomponent and having a spectral transmission characteristic such thatlight having a wavelength within a predefined waveband passes throughthe filter segment, while light having a wavelength outside of thewaveband is attenuated by the filter segment.
 7. The imaging system ofclaim 1, wherein the light that has passed through the collection lensis dispersed in a plane that is orthogonal to a direction of therelative movement between the object and the imaging system.
 8. Theimaging system of claim 1, wherein the image of the object produced bythe imaging lens moves across the TDI detector in correspondence withthe relative movement between the object and the imaging system.
 9. Theimaging system of claim 1, wherein the light from the object comprisesan unstimulated emission from the object.
 10. The imaging system ofclaim 1, further comprising a light source that is disposed to providean incident light that illuminates the object.
 11. The imaging system ofclaim 10, wherein the object scatters the incident light producingscattered light, the scattered light at least in part passing throughthe collection lens.
 12. The imaging system of claim 10, wherein theincident light illuminating the object stimulates the object to emitlight that at least in part passes through the collection lens.
 13. Theimaging system of claim 10, wherein the incident light is at leastpartially absorbed by the object, so that the light passing through thecollection lens does not include a portion of the light absorbed by theobject.
 14. The imaging system of claim 10, wherein the incident lightis reflected from the object toward the collection lens.
 15. The imagingsystem of claim 10, wherein the light source comprises at least one of:(a) a coherent light source; (b) a non-coherent light source; (c) apulsed light source; and (d) a continuous light source.
 16. The imagingsystem of claim 1, wherein the object is entrained within a fluid streamthat moves the object past the collection lens.
 17. The imaging systemof claim 1, wherein the object is carried on a support past thecollection lens.
 18. The imaging system of claim 1, wherein the TDIdetector responds to the image of the object by producing a signal thatpropagates through the TDI detector.
 19. The imaging system of claim 18,wherein a propagation rate of the signal through the TDI detector issynchronized with a motion of the image of the object on the TDIdetector as a result of the relative movement between the object and theimaging system.
 20. The imaging system of claim 18, wherein apropagation rate of the signal through the TDI detector is notsynchronized with a motion of the image of the object on the TDIdetector as a result of the relative movement between the object and theimaging system.
 21. The imaging system of claim 1, further comprising anobjective lens disposed between the object and the collection lens, saidobjective lens having a focal point at which the object is imaged; andan optical slit aligned with a direction of the relative movementbetween the object and the imaging system and disposed between theobjective lens and the collection lens at the focal point of theobjective lens, said slit substantially preventing extraneous light fromreaching the collection lens by transmitting to the collection lens thelight from the object that is focussed on the slit by the objectivelens.
 22. A method for determining one or more characteristics of amoving object from an image of the object, while there is relativemovement between the object and the imaging system, comprising the stepsof: (a) focussing light from the object along a collection path that isin a different direction than the relative movement between the objectand the imaging system; (b) dispersing the light that is traveling alongthe collection path into a plurality of light beams; (c) focussing eachof the light beams to produce a respective image corresponding to thatlight beam; (d) providing a time delay integration (TDI) detectordisposed to receive the respective images; and (e) analyzing an outputsignal from the TDI detector to determine at least one characteristic ofthe object.
 23. The method of claim 22, wherein the light is spectrallydispersed by a plurality of beam splitters arranged to reflect light ofpredetermined spectral characteristics at different predefined angles,all light from each beam splitter corresponding to a separate lightbeam, each light beam leaving the spectral dispersing component at anominal angle.
 24. The method of claim 22, wherein the light isdispersed as a function of the polarization characteristics of the lightby a plurality of beam splitters arranged to reflect light of differentpolarization characteristics at different predefined angles, all lightof each polarization characteristic corresponding to a separate lightbeam that leaves the dispersing component at a nominal angle.
 25. Themethod of claim 22, wherein the image of the object produced by the stepof focussing moves across the TDI detector, while the relative movementbetween the object and the imaging system occurs.
 26. The method ofclaim 22, wherein the light from the object comprises an unstimulatedemission from the object.
 27. The method of claim 22, further comprisingthe step of providing a filter comprising a plurality of differentspectral bandpass regions, each spectral bandpass region enablingwavelengths of light within a predefined bandpass to pass through whileattenuating wavelengths of light that are outside of the predefinedbandpass region.
 28. The method of claim 22, further comprising thesteps of: (a) providing a light source; and (b) illuminating the objectwith incident light from the light source while the object is moving.29. The method of claim 28, wherein the object scatters the incidentlight, said light that is scattered from the object at least in partpassing through the collection lens.
 30. The method of claim 28, whereinthe incident light illuminating the object stimulates the object to emitthe light that is focussed along the collection path.
 31. The method ofclaim 28, wherein the incident light is at least partially absorbed bythe object, so that the light that is focussed along the collection pathdoes not include light absorbed by the object.
 32. The method of claim28, wherein the light focussed along the collection path is incidentlight produced by the light source that has been reflected from theobject.
 33. The method of claim 28, wherein the light source comprisesat least one of: (a) a coherent light source; (b) a non-coherent lightsource; (c) a pulsed light source; and (d) a continuous light source.34. The method of claim 22, further comprising the step of entrainingthe object within a fluid stream that moves the object.
 35. The methodof claim 22, further comprising the step of carrying the object on asubstrate during the step of focussing the light from the object alongthe collection path.
 36. The method of claim 22, wherein the TDIdetector responds to the image of the object by producing a signal thatpropagates through the TDI detector.
 37. The method of claim 36, furthercomprising the step of synchronizing a motion of the image of the objecton the TDI detector with a propagation rate of the signal through theTDI detector, while the image of the object moves over the TDI detector.38. The method of claim 36, further comprising the step ofdesynchronizing a motion of the image of the object on the TDI detectorwith a propagation rate of the signal through the TDI detector, whilethe image of the object moves over the TDI detector.
 39. The method ofclaim 22, further comprising the step of preventing extraneous lightfrom reaching the TDI detector by transmitting substantially only thelight from the object along the collection path.